Pursuant to the isolation of complementary cDNA clones encoding the estrogen and other steroid receptors, a series of expression studies using genetically engineered deletion and insertion mutants have established a model for the overall organization of those biologically important regulatory molecules. Beyond defining which regions of these receptors appear to be responsible for their ligand-binding, DNA-binding and transcriptional activation functions, these studies have not, however, proven particularly insightful with regards to structural features of these protein domains which enable them to perform their respective functions. The experiments described below, which focus on the hormone-binding activity of the human estrogen receptors, will serve to address this question. A systematic mutational analysis designed to introduce amino acid substitution-mutations throughout the carboxy terminal half of the receptor is proposed to generate refined functional data on the estrogen binding domain. This will include oligonucleotide-directed mutations targeted to residues inferred to play crucial structural or potential regulatory roles. A nonbiased strategy of chemical mutagenesis will also be undertaken to overcome limitations in predicting determinants or receptor structure. Complementing these functional studies, a series of biochemical studies directly probing ligand-protein interactions and topological features of the estrogen-binding domain will explot the unique advantages of in vitro and in vivo receptor expression systems. Such a strategy is intended to build a foundation of basic structural information in the light of which emerging functional data can be more reliably interpreted. By broadening our understanding of the structural determinants which influence the affinity of this receptor for estrogen analogues and which are responsible for the antagonist activities of anti-estrogens, and underlying justification for these experiments is to assist the development of improved estrogen antagonists for use in the clinical therapy of estrogen-dependent tumors.